Inflammatory bowel disease (IBD) is characterized by relapsing intestinal inflammation and altered epithelial permeability resulting in fluid/electrolyte loss and systemic exposure to luminal antigens. Permeability changes have, in turn, been attributed to defective tight junction (TJ) structure/function. Details of epithelial TJ composition and its relationship to gate/fence function are still rudimentary. We have recently shown enrichment TJ proteins in "raft" like membrane microdomains. The major objective of this proposal is to define functionally relevant structural elements in TJs of epithelial cells with the following two specific aims. Specific Aim 1: To identify novel intercellular junction proteins involved in regulation of intestinal epithelial permeability using a monoclonal antibody approach. We have utilized TJ-enriched membrane fractions to generate four monoclonal antibodies that recognize unique epitopes in TJs of epithelial cell lines and native intestinal epithelial cells. The primary focus will be on defining the antigens for these antibodies. Epitope modulation by cytokines (IFN-gamma, HGF) important in inflammation/repair will be determined. Expression of their respective antigens in native normal and inflamed intestinal tissues will be analyzed. Specific Aim 2: To define molecular targets for tight junction proteins key in regulating intestinal epithelial permeability using a bait-peptide approach. We will capture protein components in the TJ complex using novel bait peptides representing segments of the TJ transmembrane protein, occludin. Biotinylated, photoactive bait peptides have been generated to recapitulate: 1) a 27 aa coiled-coil region in the cytoplasmic tail; and 2) 21 aa regions in the first and second extracellular loops. Peptide protein complexes in epithelial cells will be captured using a solid matrix of avidin. Functional consequences of peptide protein binding on TJ gate/fence function will be explored. Information from these studies should reveal important mechanistic insights into the structure/function of TJs and should provide novel insights into potential therapeutic targets for the prevention or correction of epithelial permeability defects associated with IBD.